RESEARCH STRATEGY Abstract Retrograde signaling is an essential organelles-to-nucleus communication pathway, central to maintenance of cellular homoeostasis in response to developmental and environmental inputs. We have identified the first stress-specific plastid-to-nucleus retrograde signaling metabolite, methylerythritol cyclodiphosphate (MEcPP), an isoprenoid precursor produced by the non-mevalonate methylerythritol phosphate (MEP) pathway. This is a conserved signaling pathway present in plants, pathogenic bacteria and the parasite, malaria, but absent in animals. As such, it has provided a validated target for the development of antibacterial and antimalarial agents. The long-term goal of our research is to delineate the perception and signal transduction components of MEcPP-mediated signaling cascade. This competitive renewal proposal specifically aims at delineating: 1) Cellular components of the MEcPP retrograde signaling pathway. To identify the components responsible for the perception and transduction of the MEcPP signal, we will initially characterize the mutated genes in the two classes of suppressor lines that, despite their equally constitutively elevated MEcPP levels, are either almost fully or only partially incapable of inducing MEcPP response genes. Systematic characterization of these perception/signaling network components will provide fundamental information regarding the underlying molecular mechanisms that link a single retrograde signaling molecule, MEcPP, to its targeted ensemble of genes. 2). Molecular link between MEcPP and unfolded protein response in the ER. Combined global transcriptome and proteome profiling, together with molecular genetics and pharmacological approaches, has enabled us to identify MEcPP as an inducer of the Unfolded Protein Response (UPR) in the endoplasmic reticulum (ER). We will decipher the MEcPP mechanism of action in induction of the UPR, and conversely delineate the role of the UPR in MEcPP-mediated responses. This finding ushers novel insight into uncharted territories of interorganellar signaling, and will provide a deeper understanding of key role of UPR in controlling principal mechanisms supporting robustness. 3). Governing components of stromule formation Imaging analyses of plants expressing a construct containing three chimeric marker genes that label plastids with CFP, ER with YFP, and the nucleus with mCherry, has established a direct correlation between enhanced MEcPP levels and increased presence of induced dynamic chloroplast tubular extensions called stromules?, that extend from plastids to ER to nucleus. We will perform comparative proteomic analyses of genotypes with varying numbers of stromules to identify cellular machineries responsible for their formation and to test the potential role of stromules as communication conduits.